THE PROTECTIVE ACTION OF THYMOL AGAINST CARBON TETRACHLORIDE HEPATOTOXICITY IN MICE

Pharmacological Research, Vol. 40, No. 2, 1999 Article No. phrs.1999.0472, available online at http:rrwww.idealibrary.com on

THE PROTECTIVE ACTION OF THYMOL AGAINST CARBON TETRACHLORIDE HEPATOTOXICITY IN MICE K. ALAM a,U , M.N. NAGI a , O.A. BADARY a , O.A. AL-SHABANAH a , A.C. AL-RIKABI b and A.M. AL-BEKAIRI a a

Department of Pharmacology, College of Pharmacy, King Saud Uni¨ ersity, P.O. Box 2457, Riyadh 11451, Saudi Arabia and bDepartment of Pathology, College of Medicine, King Saud Uni¨ ersity, Riyadh, Saudi Arabia Accepted 29 January 1999

The protective action of thymol Žparamethyl-isopropyl-phenol. was investigated against carbon tetrachloride ŽCCl 4 .-induced hepatotoxicity in male Swiss albino mice. The CCl 4 at a dose of 20 m l kgy1 produced damage to liver cells and was followed by the significant increase Ž P- 0.001. in serum alanine aminotransferase ŽALT. activity and hepatic lipid peroxidation after 24 h. The hepatocellular necrosis was further confirmed by histopathological examination of liver section. Oral administration of thymol in a single dose Ž300 mg kgy1 . resulted in significant Ž P- 0.05. amelioration of CCl 4-induced hepatotoxicity. Thymol also inhibited lipid peroxidation induced by CCl 4 in ¨ i¨ o. The protection offered by thymol was also evident from histopathology photomicrograph. In a separate in ¨ itro assay, thymol inhibited the non-enzymatic lipid peroxidation of normal mice liver homogenate induced by Fe 3q-ascorbate. The present study suggests that thymol protects the liver against CCl 4-induced toxicity and the protection may be mediated through its ability to inhibit lipid peroxidation. However, other interactions between thymol and CCl 4 remains to be elucidated. Q 1999 Academic Press KEY

WORDS:

thymol, carbon tetrachloride, hepatotoxicity, lipid peroxides.

INTRODUCTION The hepatotoxic effect of carbon tetrachloride ŽCCl 4 . is thought to result from its reductive dehalogenation by cytochrome P-450 into the trichloromethyl free radical w1, 2x. This radical quickly adds molecular oxygen to form the trichloromethyl peroxyl radical. Abstraction of hydrogen atoms from unsaturated lipids by such radicals creates carbon-centred lipid radicals w3x. These lipid radicals thereby initiate the process of lipid peroxidation. Recently, there has been interest to screen naturally occurring hepatoprotective agents. Administration of silymarin, an extract from milk thistle Silybum marianum ŽL.. w4x, Schisandrin B, the main constituent of Schisandra chinensis fruit w5x and thymoquinone, the main constituent of the volatile oil of Nigella sati¨ a ŽL.. seeds w6x have been shown to protect against CCl 4-induced hepatotoxicity in mice. U

Corresponding author.

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Thymol Žparamethyl-isopropyl-phenol. is the main constituent of the essential oils of Thymus ¨ ulgaris ŽL.. w7x. It possess antioxidant properties and has been suggested as a natural replacement for synthetic antioxidant additives w8x. In the present study, we investigated thymol for its anti-hepatotoxic action against CCl 4-induced liver injury by means of biochemical markers and histopathology. Also, the effect of thymol on the non-enzymatic lipid peroxidation in normal mouse liver homogenate, induced in ¨ itro by Fe 3q-ascorbate, was evaluated and compared with butylated hydroxytoluene, a synthetic antioxidant.

MATERIALS AND METHODS

Chemicals Thymol Žparamethyl-isopropyl-phenol. and carbon tetrachloride ŽCCl 4 . were purchased from Koch Light Laboratories Ltd., England. Thiobarbituric acid ŽTBA. and butylated hydroxytoluene ŽBHT. Q 1999 Academic Press

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were purchased from Fluka, Switzerland. All other chemicals and reagents were of highest grade commercially available.

Animals Male Swiss albino mice weighing 25]27 g were obtained from the Breeding Centre at King Saud University, Riyadh, Saudi Arabia. The animals were kept in a controlled condition of temperature 22 " 18C, relative humidity Ž50]55%. and 12 h]12 h light]dark cycle. The mice had free access to standard rodent food ŽPurina chow. and water, and were housed for 72 h before use.

Experimental design

Carbon tetrachloride Ž5]40 m l kgy1 . was administered intraperitoneally Ži.p.. into the mice in 0.2 ml corn oil. After 24 h, blood was collected from the orbital sinus under light ether anesthesia and animals were killed by cervical dislocation. Thymol Ž50]300 mg kgy1 , p.o.. was given in corn oil immediately before the CCl 4 injections. The control mice received the vehicle Žcorn oil; 10 mg kgy1 . or thymol Ž300 mg kgy1 ..

Enzyme assay Serum alanine aminotransferase ŽALT. was determined kinetically using commercial kit ŽBiosystems, Barcelona, Spain.. Previous studies w6, 9x demonstrated that CCl 4 gives maximum ALT response after 24 h; we used this time period in the present study.

Lipid peroxidation Thiobarbituric acid reactive substances ŽTBARS., measured as malonaldehyde ŽMDA., were determined in liver homogenates by the method described earlier w10x and was considered as an index of lipid peroxidation.

Assay of non-enzymatic lipid peroxidation in vitro Lipid peroxidation was carried out as described

Table I Effects of carbon tetrachloride on serum alanine aminotransferase activity in mice CCl4 Ž m l kgy1 .

ALT ŽIU ly1 .

0 Žcorn oil. 5 10 20 40

25 " 1.1 34 " 1.9 42 " 3.7 856 " 41.6U 1754 " 61.3U

Results are expressed as mean " SEM of 10 mice. Data were analysed by one-way ANOVA followed by Dunnett’s test. CCl 4 was administered i.p. and serum ALT was measured after 24 h. U Significant increase Ž P- 0.001. compared to corn oil Žvehicle.-treated mice.

previously w11x with slight modification. The assay mixture contained varying amount of inhibitors Ž0.2]10 m M. in 10 m l ethanol, 0.75 ml phosphate buffered saline Ž50 mM, pH 7.4., 50 m l normal mice liver homogenate Ž10%. and 0.1 ml of 1 mM ferric chloride. The contents were mixed thoroughly and peroxidation was initiated by 0.1 ml of 1 mM ascorbic acid. The tubes were placed at 378C for 30 min and the extent of peroxidation was measured by TBA test. To the above tubes were added 0.1 ml BHT Ž2% wrv., 1.0 ml TBA Ž1% wrv in 0.05 M NaOH. and 1.0 ml TCA Ž2.8% wrv. and placed in water bath at 808C for 20 min. At the end of incubation the tubes were cooled and centrifuged at 3200 rev. miny1 for 5 min. The chromogen was extracted with 2.0 ml n-butanol and absorbance was read at 532 nm. Tubes without antioxidants were subjected to TBA test and served as control.

Histopathology Histological examination was performed on approximately 50% of randomized animals of each group. Liver samples were taken from the distal portion of the left lateral lobe. The tissue was fixed for at least 48 h in 10% formalin. The samples were then embedded in paraffin, cut into 5-m m sections,

Table II Effects of thymol treatment on carbon tetrachloride-induced hepatotoxicity in mice Treatment

ALT (IU l y 1)

Thymol Ž300 mg kgy1 . CCl4 Ž20 m l kgy1 . CCl4 Ž20 m l kgy1 . q thymol Ž50 mg kgy1 . CCl4 Ž20 m l kgy1 . q thymol Ž100 mg kgy1 . CCl4 Ž20 m l kgy1 . q thymol Ž200 mg kgy1 . CCl4 Ž20 m l kgy1 . q thymol Ž300 mg kgy1 .

29 " 1 1023 " 57.5 977 " 61 570 " 45.8 404 " 28.1 246 " 16.4U

Results are expressed as mean " SEM of 10 mice and data were analysed by one-way ANOVA followed by Dunnett’s test. Thymol was administered Žp.o.. immediately before CCl 4 injection Ži.p... Serum ALT was measured 24 h after CCl 4 treatment. U Significant decrease Ž P- 0.05. compared to mice treated with CCl 4 alone.

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and stained with haematoxylin and eosin for examination by light micrography.

Statistics Results are expressed as mean " SEM. Data were compared by ANOVA followed by Dunnett’s t-test and Tukey]Kramer multiple comparisons test; all differences were considered significant when P0.05.

Results

The effects of different doses of CCl 4 Ž5]40 m l kgy1 , i.p.. on serum ALT activity in mice are summarised in Table I. Carbon tetrachloride at a dose of 20 m l kgy1 and 40 m l kgy1 produced significant increase Ž P- 0.001. in ALT activity compared to vehicle Žcorn oil.-treated mice. The increase in ALT activity by CCl 4 Ž20 m l kgy1 . was sub-maximal. Carbon tetrachloride Ž20 m l kgy1 , i.p.. was co-administered with different doses of thymol Ž50]300 mg kgy1 .. Co-treatment with thymol reduced ALT activity induced by carbon tetrachloride ŽTable II.. A significant decrease Ž P- 0.05. in enzyme activity was observed with 300 mg kgy1 of thymol. Similarly, hepatic lipid peroxidation was significantly Ž P0.001. decreased ŽFig. 1. in the presence of thymol compared to mice treated with CCl 4 alone. Thymol Ž300 mg kgy1 . alone had no effect on serum ALT activity. Histopathological examination of the liver sections of the mice treated with CCl 4 Ž20 m l kgy1 . showed multiple centrilobular hepatocellular necrosis wFig. 2Ža.x. Mice simultaneously treated with thymol Ž300 mg kgy1 . and CCl 4 showed no signs of hepatocellular necrosis wFig. 2Žb.x. However, there

161

was a mild to moderate centrilobular inflammatory cell infiltration with some hepatocellular swelling. Mice treated with thymol alone had normal liver histology Žfigure not shown.. Fig. 3 shows the effect of thymol and BHT Žstandard antioxidant. on Fe 3qascorbate-induced lipid peroxidation in normal mice liver homogenate in ¨ itro. Like BHT, thymol also inhibited the lipid peroxidation but to a lesser extent. The apparent IC 50 Žinhibitor concentration for 50% inhibition. for thymol and BHT were calculated to be 7.5 m M and 0.75 m M, respectively.

DISCUSSION The results of the study demonstrated that the thymol decreases the CCl 4-induced liver injury in mice, as evident from the significant decrease in serum ALT activity and lipid peroxidation. The biochemical mechanism involved in the development of CCl 4 hepatotoxicity are well documented. It is now believed that the formation of reactive trichloromethyl radicals from CCl 4 metabolism is the crucial factor in the pathogenesis of CCl 4 hepatotoxicity w12x. The findings that CCl 4 toxicity could be ameliorated by pretreatment with inhibitors of CCl 4 metabolism w13x and antioxidants w6, 14, 15x support this notion. Moreover, lipid peroxidation was suggested as the major molecular mechanism involved in CCl 4 toxicity w16x, the first morphologic and functional alterations occurring in the membrane of endoplasmic reticulum w17x. In this connection thymol acted as a free radical scavenger of lipid peroxidation in ¨ itro ŽFig. 3.. This finding supports

Fig. 1. Effect of thymol on CCl 4-induced lipid peroxidation in mice liver homogenate. Results are expressed as mean q SEM Ž n s 8.. Thymol Ž300 mg kgy1, p.o.. was given immediately before CCl 4 injections Ž20 m l kgy1, i.p.. TBARS were determined after 24 h of CCl 4 administration and data were analysed by one-way ANOVA followed by Tukey]Kramer U multiple comparisons test. Significant increase Ž P- 0.001. compared to corn oil Žvehicle.-treated mice. aSignificant decrease Ž P- 0.001. compared to mice treated with CCl 4 alone.

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Fig. 2. Light micrographs of mice liver treated with CCl 4 , without and with thymol. Representative sections from Ža. mouse treated with CCl 4 Ž20 m l kgy1, i.p.., showing extensive centrilobular hepatocellular necrosis; and Žb. mouse treated with CCl 4 Ž20 m l kgy1, i.p.. and thymol Ž300 mg kgy1, p.o.. showing absence of hepatocellular necrosis. Liver samples were taken 24 h after CCl 4 injection. Haematoxylin and eosin stain ŽHrE., magnification =250.

the earlier observations that thymol acts as a good scavenger of peroxyl radical w8x. In summary, this study suggests that the oral administration of thymol significantly ameliorates CCl 4 hepatotoxicity in mice. The compound may be protecting the liver by preventing peroxidation of lipids of the endoplasmic reticulum. However, the possibility that thymol might suppress the cytochrome P-450 mediated metabolic activation of CCl 4 itself can not be ruled out.

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Fig. 3. Inhibition of non-enzymatic lipid peroxidation in ¨ itro by thymol Žfilled circle. and BHT Žopen circle., a standard antioxidant. Each point represents the mean of three tests Žfor clarity, SEM bars are not shown; values were - 10% of the mean..

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